Identification of miR-146a target genes in mouse monocyte subsets

To gain insight into the mechanisms underlying miR-146a-mediated modulation of Ly6Chigh monocyte function, we compared the expression profiles of Ly6Chigh and Ly6Clow monocytes in miR-146a+/+ (WT) versus miR-146a-/- (KO) conditions. Ly6Clow and Ly6Chigh monocyte subsets were isolated from spleen samples of miR-146a-/- mice or control littermates using a FACSAria sorter, and total RNA of each cell subsets were extracted using miRNeasy mini kit (Qiagen). The integrity of isolated and pooled RNA was assessed for each sample using an Agilent 2100 Bioanalyzer (Agilent, Waldbronn, Germany) and amount was checked with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE). The generation of biotinylated anti-sense cRNA was performed with 100 ng of total RNA using the Affymetrix IVT Express protocol (3' IVT Express 2010 technical manual P/N 702646 Rev8). Following fragmentation, 15 µg of the cRNA were hybridized for 16 h at 45 °C on GeneChip Mouse 430_2 arrays in the Affymetrix oven 645. GeneChips were washed and stained in the Affymetrix Fluidic station 450 with HWS kit. Chips were scanned using the Affymetrix GeneChip scanner 3000 7G, and the raw data were generated with the Affymetrix Expression Console software v 1.2.1. Raw data were analyzed with High Performance Chip Data Analysis (HPCDA) and database BioRetis using one vs. one chip analyses.

为深入解析miR-146a介导的Ly6C高表达单核细胞功能调控机制，我们对比了miR-146a+/+（野生型，Wild Type，WT）与miR-146a-/-（基因敲除，Knock Out，KO）条件下Ly6C高表达与Ly6C低表达单核细胞的表达谱。我们从miR-146a基因敲除小鼠及其同窝野生型对照的脾脏样本中分离得到两类单核细胞亚群，采用FACSAria流式细胞分选仪完成分选；随后使用miRNeasy Mini试剂盒（Qiagen公司）提取各亚群的总RNA。针对每份样本，我们采用Agilent 2100生物分析仪（Agilent，德国瓦尔德布龙）评估所提取总RNA的完整性，并通过NanoDrop ND-1000分光光度计（NanoDrop Technologies，美国特拉华州威尔明顿）检测RNA的总含量。我们以100 ng总RNA为起始模板，依照Affymetrix IVT Express实验流程（《3' IVT Express 2010技术手册》P/N 702646 Rev8）制备生物素标记的反义cRNA。经片段化处理后，取15 µg cRNA置于Affymetrix 645型杂交炉中，与GeneChip Mouse 430_2芯片在45℃条件下杂交16小时。随后将基因芯片放置于Affymetrix Fluidic Station 450型流体工作站中，搭配HWS试剂盒完成洗涤与染色步骤；之后使用Affymetrix GeneChip Scanner 3000 7G型扫描仪对芯片进行扫描，并通过Affymetrix Expression Console软件v1.2.1生成原始数据。我们采用高性能芯片数据分析工具（High Performance Chip Data Analysis, HPCDA）与BioRetis数据库，通过单芯片两两比较分析对原始数据进行处理。